Periodic Reporting for period 1 - EpiSeq (Single molecule sequencing and biophysical properties of oxidized genomic DNA using magnetic tweezers.)
Reporting period: 2019-07-01 to 2021-06-30
Oxidative stress is a persistent threat to genomic DNA and RNA and is associated with major causes of human mortality, including cancers, diabetes and aging. 8-oxoguanine (8-oxoG) is the most common product of oxidative stress in nucleic acids. Evidence has accumulated to show that 8-oxoG is not only a pre-mutagenic DNA base lesion but also has essential roles in the regulation of gene expression and may act as an epigenetic mark. For such functions, the oxidative modification would need to occur in regulatory regions of genome such as promoters. Indeed, in vitro and genome-wide studies of the distribution of 8-oxoG showed that potential G4-forming sequences (repetitive guanine-rich sequences which can form G-quadruplex (G4) secondary structures), presented in ~50% promoters of human genes, are preferential substrates for oxidation. However, the mechanism of how 8-oxoG regulates gene transcription remains unclear. In addition, some recent studies showed contradicting conclusions concerning the distribution of 8-oxoG at gene regulatory regions, which makes the role of 8-oxoG in gene regulation an intense area of research.
The main objectives of this project are:
1) study in vitro the effect of 8-oxoG modification on G-quadruplex (G4) folding at biochemical and biophysical level
2) develop a reliable method to capture a specific genomic fragment, starting from yeast genomic DNA
3) develop the single-molecule technology to detect the presence and location of oxidize guanines in the DNA molecules, at single-nucleotide resolution
The main objectives of this project are:
1) study in vitro the effect of 8-oxoG modification on G-quadruplex (G4) folding at biochemical and biophysical level
2) develop a reliable method to capture a specific genomic fragment, starting from yeast genomic DNA
3) develop the single-molecule technology to detect the presence and location of oxidize guanines in the DNA molecules, at single-nucleotide resolution
Objective 1:
To study in vitro the interplay between 8-oxoG, DNA topology (such as G-quadruplex or G4) and protein interactions to modulate gene expression, we have developed a label-free single-molecule assay to follow the dynamics of G4s and their interaction with proteins in double-stranded DNA (dsDNA) to get closer to their biological context (Tran PLT et al, Nucleic Acids Research, 2021 and Valle-Orero J, Tran PLT, et al, in preparation).
In our recent publication (Tran PLT, et al, Nucleic Acids Research, 2021), we have shown that G4s are much more persistent in the promoters and the replication origin than in human telomere repeats. In addition, we revealed that commonly used G4-specific-ligands and antibodies increase both the folding rate and the persistence of the G4 structures in this context. Our novel assay opens new perspectives for the measurement of G4 dynamics in dsDNA, which is critical to understand the role of G4 in gene regulation.
Besides their regulatory function, persistent G4s can be a roadblock for replication and transcription. Using magnetic tweezer system, we manage to visualize in real-time and calculate the time (~15s) for G4 structure resolution by Pif1 helicase (Valle-Orero J, Tran PLT, et al, in preparation).
Using the recently developed assays, we studied the effect of oxidative lesions on G4 formation and how 8-oxoG can affect protein binding. We showed that 8-oxoG disturbed G4-cMYC folding differently according to the position of the modification (Tran PLT et al., in preparation). Next, we will study how these modifications could favor or disturb protein binding (such as transcription factors or base excision repair (BER) enzymes). These results will enable to better understand the mechanism of how 8-oxoG modulates gene expression (its epigenetic function).
Objective 2:
To capture a specific genomic DNA fragment from yeast, we have designed and assembled in vitro CRISPR/Cas9 complexes guided to specifically cut away a given locus from the rest of the genome. We have successfully captured the inserted CEB25 fragment (a synthetic G-rich DNA fragment mimicking a G-rich human minisatellite repeat) from yeast genomic DNA and ligated it to the adapters to manipulate it under magnetic tweezers. Next, we will optimize it for higher organisms, including the human genome.
Objective 3:
We have already screened all existing commercial 8-oxoG antibodies for this objective and we have selected the best one that gives accurate results. 8-oxoG, as well as other modifications, such as 5-methyl cytosines or 6-methyl adenines, can be systematically and unambiguously identified on synthetic DNA molecules at single-nucleotide resolution using SIMDEQ (SIngle Molecule DEtection and Quantification) sequencing platforms (from Depixus) based on magnetic tweezers (Wang Z et al., Communications Biology, 2021). Next, we will use this technology to map genome-wide 8-oxoG at single-nucleotide resolution.
To study in vitro the interplay between 8-oxoG, DNA topology (such as G-quadruplex or G4) and protein interactions to modulate gene expression, we have developed a label-free single-molecule assay to follow the dynamics of G4s and their interaction with proteins in double-stranded DNA (dsDNA) to get closer to their biological context (Tran PLT et al, Nucleic Acids Research, 2021 and Valle-Orero J, Tran PLT, et al, in preparation).
In our recent publication (Tran PLT, et al, Nucleic Acids Research, 2021), we have shown that G4s are much more persistent in the promoters and the replication origin than in human telomere repeats. In addition, we revealed that commonly used G4-specific-ligands and antibodies increase both the folding rate and the persistence of the G4 structures in this context. Our novel assay opens new perspectives for the measurement of G4 dynamics in dsDNA, which is critical to understand the role of G4 in gene regulation.
Besides their regulatory function, persistent G4s can be a roadblock for replication and transcription. Using magnetic tweezer system, we manage to visualize in real-time and calculate the time (~15s) for G4 structure resolution by Pif1 helicase (Valle-Orero J, Tran PLT, et al, in preparation).
Using the recently developed assays, we studied the effect of oxidative lesions on G4 formation and how 8-oxoG can affect protein binding. We showed that 8-oxoG disturbed G4-cMYC folding differently according to the position of the modification (Tran PLT et al., in preparation). Next, we will study how these modifications could favor or disturb protein binding (such as transcription factors or base excision repair (BER) enzymes). These results will enable to better understand the mechanism of how 8-oxoG modulates gene expression (its epigenetic function).
Objective 2:
To capture a specific genomic DNA fragment from yeast, we have designed and assembled in vitro CRISPR/Cas9 complexes guided to specifically cut away a given locus from the rest of the genome. We have successfully captured the inserted CEB25 fragment (a synthetic G-rich DNA fragment mimicking a G-rich human minisatellite repeat) from yeast genomic DNA and ligated it to the adapters to manipulate it under magnetic tweezers. Next, we will optimize it for higher organisms, including the human genome.
Objective 3:
We have already screened all existing commercial 8-oxoG antibodies for this objective and we have selected the best one that gives accurate results. 8-oxoG, as well as other modifications, such as 5-methyl cytosines or 6-methyl adenines, can be systematically and unambiguously identified on synthetic DNA molecules at single-nucleotide resolution using SIMDEQ (SIngle Molecule DEtection and Quantification) sequencing platforms (from Depixus) based on magnetic tweezers (Wang Z et al., Communications Biology, 2021). Next, we will use this technology to map genome-wide 8-oxoG at single-nucleotide resolution.
This interdisciplinary project contributes first to the development of a label-free, cost-effective and accurate sequencing technique to map genome-wide 8-oxoG at single-nucleotide resolution. Second, these results enable to study the mechanism of how 8-oxoG modulates gene expression. The data on genome-wide distribution of 8-oxoG as well as on the interplay between 8-oxoG and gene transcription through G4 regulation will contribute to better understand the biological function and the role of 8-oxoG in different diseases (e.g. cancers, neurodegenerative disorders, diabetes and cardiovascular diseases).
In contrast to most of the current sequencing techniques, the technology used in this study does not require a PCR amplification step, and in addition, the process of hairpin molecule preparation is non-destructive allowing to keep intact the native genomic DNA. Thus, the same molecules can be used and re-used many times, enabling a highly precise detection of different base modifications through repeated interrogation.
This hands-on exposure to a breakthrough epigenetic sequencing technology (the 4th generation of Next Generation Sequencing) will be useful for our future research, at a prime time when it has received wide support from industrial investment (to Depixus). In addition, the genomics of 8-oxoG and its role as an epigenetic mark is a continuously growing and dynamic field. These novel and original assays have brought and will bring in many more publications and collaborators to the host organization.
In contrast to most of the current sequencing techniques, the technology used in this study does not require a PCR amplification step, and in addition, the process of hairpin molecule preparation is non-destructive allowing to keep intact the native genomic DNA. Thus, the same molecules can be used and re-used many times, enabling a highly precise detection of different base modifications through repeated interrogation.
This hands-on exposure to a breakthrough epigenetic sequencing technology (the 4th generation of Next Generation Sequencing) will be useful for our future research, at a prime time when it has received wide support from industrial investment (to Depixus). In addition, the genomics of 8-oxoG and its role as an epigenetic mark is a continuously growing and dynamic field. These novel and original assays have brought and will bring in many more publications and collaborators to the host organization.